Novel Phosphorylation Sites in the <i>S. cerevisiae</i> Cdc13 Protein Reveal New Targets for Telomere Length Regulation

The <i>S. cerevisiae</i> Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the <i>cdc13-2</i> allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of <i>in vivo</i> phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two <i>in vitro</i> targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. Multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics

Novel Phosphorylation Sites in the <i>S. cerevisiae</i> Cdc13 Protein Reveal New Targets for Telomere Length Regulation

The <i>S. cerevisiae</i> Cdc13 is a multifunctional protein with key roles in regulation of telomerase, telomere end protection, and conventional telomere replication, all of which are cell cycle-regulated processes. Given that phosphorylation is a key mechanism for regulating protein function, we identified sites of phosphorylation using nano liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS). We also determined phosphorylation abundance on both wild type (WT) and a telomerase deficient form of Cdc13, encoded by the <i>cdc13-2</i> allele, in both G1 phase cells, when telomerase is not active, and G2/M phase cells, when it is. We identified 21 sites of <i>in vivo</i> phosphorylation, of which only five had been reported previously. In contrast, phosphorylation of two <i>in vitro</i> targets of the ATM-like Tel1 kinase, S249 and S255, was not detected. This result helps resolve conflicting data on the importance of phosphorylation of these residues in telomerase recruitment. Multiple residues showed differences in their cell cycle pattern of modification. For example, phosphorylation of S314 was significantly higher in the G2/M compared to the G1 phase and in WT versus mutant Cdc13, and a S314D mutation negatively affected telomere length. Our findings provide new targets in a key telomerase regulatory protein for modulation of telomere dynamics

PILOT_PROTEIN: Identification of Unmodified and Modified Proteins via High-Resolution Mass Spectrometry and Mixed-Integer Linear Optimization

A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized. A sequence tag procedure is then incorporated along with the untargeted PTM identification algorithm, PILOT_PTM, to determine a list of all modification types and sites for each protein. The unmodified protein identification algorithm, PILOT_PROTEIN, is compared to the methods SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both prepared protein samples and a more complex chromatin digest. The algorithm demonstrates superior protein identification accuracy with a lower false positive rate. All materials are freely available to the scientific community at http://pumpd.princeton.edu

On the Histone Lysine Methyltransferase Activity of Fungal Metabolite Chaetocin

Histone lysine methyltransferases (HKMTs) are an important class of targets for epigenetic therapy. <b>1</b> (chaetocin), an epidithiodiketopiperazine (ETP) natural product, has been reported to be a specific inhibitor of the SU­(VAR)­3-9 class of HKMTs. We have studied the inhibition of the HKMT G9a by <b>1</b> and functionally related analogues. Our results reveal that only the structurally unique ETP core is required for inhibition, and such inhibition is time-dependent and irreversible (in the absence of DTT), ultimately resulting in protein denaturation. Mass spectrometric data provide a molecular basis for this effect, demonstrating covalent adduct formation between <b>1</b> and the protein. This provides a potential rationale for the selectivity observed in the inhibition of a variety of HKMTs by <b>1</b> in vitro and has implications for the activity of ETPs against these important epigenetic targets